1. Field of the Invention
The present invention relates to a method and apparatus for assessing the characteristic response of a medium to an excitation transient which causes the medium to emit a series of signals over a period of time which is long compared to the duration of the excitation transient.
The present invention is particularly applicable to the assessment of a fluorescent decay but could be applied to any circumstances in which an excitation triggers a response that has decay or other time dependent characteristics which continue for a substantial period of time after termination of the excitation. The nature of the excitation may be the same as or different from the nature of the emission, for example light trigger and light emission or chemical trigger and light emission, and the excited species need not be excited directly, for example excimer and fluorescence energy transfer where the excited species is not the emitting species.
2. Description of Related Art
In conventional fluorescence decay analysis, in which a series of photons are emitted after pulse excitation, the time interval between the excitation pulse and the first emitted photon is monitored. The sample is repeatedly excited to enable the accumulation of data representing the distribution of the arrival times of the first photons resulting from each excitation. A significant delay is required between successive excitations to ensure that aliasing does not occur, that is to ensure that a photon emission resulting from one excitation is not detected as the first emitted photon resulted from the next excitation. Typically the delay required is from 10 to 100 times the fluorescent lifetime. These delays, coupled with the need for repeated excitation, prevent high speed measurements being obtained.
A further disadvantage of conventional fluorescence detection methods is that the intensity of successive excitation pulses may vary. This variation of excitation pulse intensity will cause distortion of the measured fluorescent lifetime. The distortion is particularly pronounced if the excitation mechanism is non linear, for example two-photon absorption.
The distortion may be reduced by stabilising the source of the excitation pulses. However, such stabilisation will not fully remove intensity variation between successive excitation pulses.
The effect of excitation pulse intensity variation is often reduced using normalisation. Normalisation involves modifying the statistical significance of a detected fluorescence photon according to the intensity of the corresponding excitation pulse. A disadvantage of normalisation is that it requires high speed electronics. Furthermore, where the normalisation involves recording the intensity of the excitation pulse, the amount of data which must be stored during a measurement is significantly increased. This is because the intensity of the excitation pulse will typically be represented by 12 or more bits, whereas the detected fluorescence photon be represented by a single bit.
It is an object of the present invention to obviate or mitigate at least some of the problems outlined above.
According to the present invention, there is provided a method for assessing the characteristic response of a medium to an excitation transient of predetermined duration which causes the medium to emit a series of signals over a period of time which is long relative to the duration of the excitation transient, wherein the signals are detected, the duration of each interval between successive signals is measured, and a relationship relating the interval between the excitation transient and the emission of each signal to the interval between each signal and the preceding signal in the series is derived to represent the characteristic response.
Preferably the interval between the excitation transient and the emission of each signal is plotted against the interval between each signal and the preceding signal in the series, a curve is fitted to the plot, the position of the minimum value of the interval between the excitation transient and the emission of each signal as represented by the curve is determined, and the interval between successive signals corresponding to the position of the minimum is determined to provide a measure of the characteristic response of the medium.
The excitation transient may be a short pulse (i.e. shorter than the duration of the characteristic response to be measured). Alternatively the excitation may be a long pulse which provides a steady state of excitation, the excitation transient being a rapidly decaying falling edge of the long pulse. In a further alternative, the excitation may be a two-photon excitation, obtained via two excitation sources, in which case the excitation transient is the period during which both sources excite the medium simultaneously.
The present invention makes it possible to measure for example a fluorescent decay lifetime from pulse (quantum) signals resulting from only a single excitation transient of very short duration, for example a few nanoseconds, or less.
The invention is advantageous because it reduces the effect of variation of the excitation intensity.
The invention allows the characteristic response of the medium to be assessed from a single excitation transient.
The invention also allows the measurement of a characteristic response from a series of excitations of a medium, i.e. by averaging the lifetimes measured in response to a series of excitations. Intensity variations between successive excitations will not distort the measured fluorescence decay lifetime because the measurement according to the invention is unaffected by intensity variations. Therefore, no normalization is required.
Where normalisation is used, for example where the quantity of a fluorescent medium is to be determined, the amount of data that must be stored to perform the normalisation is small because the intensity of each excitation may be used to normalise the series of detected fluorescence photons emitted in consequence of that excitation. This compares to the prior art, wherein each detected fluorescence photon must be normalised according to a separate excitation intensity measurement.
The invention is particularly applicable to the analysis of signals resulting from excitation of fluorophores. The signals may result from direct excitation or energy transfer to one species from another species excited by the excitation.
The timing of the signals may be determined from any convenient portion of each signal, for example the leading edge.
A property of the excitation may be used to normalise the detected signals.
A bleaching rate of a fluorophore may be measured. The bleaching rate may be used as a marker to monitor the conformation, environment or binding of the fluorophore.
The invention may be applied to the assessment of a large number of samples of a particular medium using a single source. Excitation may be delivered to the samples from the single source and received by a single detector. Each of the samples may receive an excitation in turn, signals from each of the samples being detected in turn, or alternatively each of the samples may receive an excitation simultaneously, with signals from all of the samples being detected in parallel. This latter approach enables rapid assessment of a large number of samples to determine whether any of them is generating a characteristic response before the application of the first approach to assess individual samples which do exhibit some characteristic response.